State of the art methods for cell sorting, which perform sorting of a heterogeneous mixture of biological cells into two or more containers based upon the specific light scattering and/or fluorescent characteristics of each cell, include fluorescence-activated cell sorting (FACS) and methods based on flow cytometry. Such sorting may consist of two phases, e.g. a first phase may be a so-called discrimination phase, in which a cell is classified based on the fluorescence and/or light scattering properties of the cell, while the second phase may be a so-called fractionation phase, in which the fluid stream is separated into charged droplets which can be deflected mechanically or electrostatically in order to divert the droplets into different bins. FACS has been the workhorse of biology researchers, which may be due to a number of reasons. For example, FACS has a high single cell level sensitivity and therefore may be capable of detecting cell surface markers at the single cell level, which may be largely due to the excellent sensitivity of the fluorescence detection. Furthermore, FACS has a high throughput of sorting and/or counting, which allows population-averaged single cell data. Today's high speed sorter systems can analyze up to 100,000 events per second. This throughput may be limited at least by the speed at which droplets can be deflected. FACS furthermore has the ability to track multiple parameters. Modern FACS instruments may have multiple lasers and detectors, adapted for versatile multispectral fluorescent staining applications. FACS systems are being used in a variety of applications discriminating cells based on size, morphology, cell pigments, protein expression level, fluorescent probes for in situ hybridization to visualize one or more specific regions of the genome inside the cell, e.g. via the so-called Flow-FISH, intracellular and nuclear protein markers, Green Fluorescent Proteins, pH, calcium stainings, etc. It may be possible to transport particular embodiments of this cell sorter between locations quickly, e.g. so that it could be a part of mobile system.
Modern FACS systems may have the disadvantage of a large equipment size and high cost, contamination between different samples, the serial nature of the sorting and low cell viability after ejection. There also exists a trade-off between the sorting speed, the purity rate and the recovery rate. Especially in modern cancer and immunology research, it may be important to recover all of the cells from the cell sorter with the highest achievable purity rate.
Microfluidic FACS systems bring miniaturization and disposability to cell sorting. Microfluidic FACS systems are generally perceived as slower than the macroscopic versions, see e.g. Nature Vol. 441, pg. 1179, but easier to parallelize. For example, IMT (Santa Barbara) has developed a rare cell purification system that uses 32 parallel channels with tiny micromechanical valves, optics and electromagnetic actuation to divert cells for rapid collection following detection of appropriate fluorescent markers. Cytonome has built an inventive microfluidic switch providing cell sorting of 2000 cells per second per channel by using 144 concurrently operating microfluidic sorters. Such a switch may be able to reach a sorting speed of 288,000 cells per second.
The United States patent application US 2008/213821 also discloses such a FACS device for sorting objects in a flowing medium. This device comprises a fluid handling unit comprising a plurality of microfluidic channels, which comprise a detection region for conducting flowing medium along a corresponding detector and a microfluidic switch arranged downstream of the detection region for controllably directing each object in the flowing medium to a plurality of outlets. Furthermore, a real-time characterization of detection signals obtained for each of the objects when passing through the detection region for controlling the microfluidic switch is described. In-flow imaging systems are also known in the art, e.g. the system disclosed by Amnis in US 2009/003681. This system uses conventional optics, e.g. a collection lens, a light dispersing element, an imaging lens and a CCD detector, to perform cell imaging in flow for diagnostic purposes. A time delay integration approach may be used for providing improved images of fast moving objects. This may result in high resolution images from which cell characteristics can be derived. For example, the Amnis system may be able to image up to 4000 cells per second.
Furthermore, a lens-free imaging system may be known, e.g. from Lab Chip, 2009, 9, 777-787. This paper discloses a lens-free holographic cytometer and an imaging and reconstruction method that results in an improvement of the reconstructed images with much richer texture information from the digitally processed holographic images. The system may be used for characterization and counting of cells statically present on a CMOS chip. This paper demonstrates that it is possible to perform identification or characterization of a heterogeneous cell solution on a chip based on pattern recognition of the holographic diffraction pattern of each cell type. The paper proposes the use these principles of lens-free imaging to do in-flow cell imaging at a very high speed.
However, the systems described above may not be appropriate for in-flow analysis and sorting of cells.
The described lens-free system is a static system for the analysis of cells. The cells are present in a micro-fluidic device; however, the cells are not in-flow. A different architecture and methodology is required to analyze cells in-flow.
The in-flow imagers described above require bulky conventional optics making them costly, expensive and not suitable for transport.
However, there remains a need for an in-flow cell analyzing/sorting system which is capable of a high throughput, which is flexible in terms of difference between cells under investigation, and which is reliable, easy-to use and also compact. Such systems with these properties are not currently available.